This invention relates to a novel nuclear hormone receptor co-activator. The invention further relates to high throughput screening assays with these receptors, as well as utilization of the co-activator for developing therapeutic measures for human diseases.
Nuclear hormone receptors are ligand-regulated transcription factors that play diverse roles in cell growth, differentiation, development, and homeostasis. The nuclear receptor superfamily has been divided into two sub-families: the steroid receptor family and the thyroid hormone/retinoid (non-steroid) receptor family (51). The steroid receptor family includes receptors for glucocorticoids (GR), mineralcorticoids (MR), progestins (PR), androgens (AR) and estrogens (ERs) (51). The non-steroid receptor family includes receptors for thyroid hormones (TRs), retinoids (RARs and RXRs), 1,25-(OH)2 vitamin D (VDR), prostanoids (PPARs) as well as many orphan receptors whose ligands (if any) remain to be defined (49, 51). Members of the nuclear receptor superfamily share common structural and functional motifs. Nevertheless, an important difference exists between the two sub-families. Steroid receptors primarily act as homodimers by binding to their cognate palindromic hormone response elements (HREs) (77, 78). In contrast, members of the non-steroid receptor family can bind to DNA as monomers, homodimers, and heterodimers (25, 78). Their corresponding HREs are also complex, and can be organized as direct repeats, inverted repeats, and everted repeats (49). Therefore, the combination of heterodimerization and HRE complexity provides the potential to generate enormous diversity in receptor-mediated regulation of target gene expression.
Structural and functional studies indicate that the ligand binding domain (LBD) of many members of the thyroid hormone/retinoid receptor family harbors diverse functions. In addition to ligand binding, the LBD also plays roles in mediating receptor dimerization, hormone-dependent transactivation, and in the case of TR and RAR, ligand-relieved gene silencing (54, 61). The carboxyl-terminal helix of the LBD has been implicated in playing an important role in ligand-dependent conformational changes and transactivation (6, 9, 21, 43). Although it has been suggested that an activation function (AF-2) resides in this C-terminal helix, recent studies indicate that AF-2 results from a ligand-induced conformational change involving diverse areas of the LBD (23, 66). Thus, ligand binding serves to switch the receptor from one functional state (e.g. inactive or silencing) to another (e.g. transactivation).
Although much has been learned from studying the structure and function of these receptors, the detailed molecular mechanism(s) of transcriptional regulation by these receptors is not well understood. Efforts to understand the molecular mechanism of transcriptional repression by unliganded TRs and RARs have led to the description (12) and isolation of putative co-repressor proteins SMRT and N-CoR, which interact with the LBD of these receptors in the absence of their ligands (15, 36). The recent discovery that both SMRT and N-CoR form complexes with Sin3 and a histone deacetylase suggests that chromatin remodeling by histone deacetylation may play a role in receptor-mediated transcriptional repression (33, 55).
In a somewhat parallel approach, the identification of co-activators has recently received extensive experimental attention in order to elucidate the molecular mechanism(s) of transcriptional activation by nuclear receptors (27). Identified co-activator proteins primarily belong to two groups: the SRC-1 family and the CBP/p300 family. The SRC-1 family includes SRC-1/NCoA-1 (37, 58, 74), and the related proteins GRIP1/TIF2/NCoA-2 (34, 35, 74, 79), and AIB1/p/CIP/ACTR/RAC3/TRAM-1 (2, 14, 44, 73, 74). The second group of co-activators includes CBP and its homolog p300, which not only influence the activity of nuclear receptors (13, 31, 37), but also functionally interact with many transcription factors such as CREB (3, 16, 40, 46), the Stats (10, 87), API (4, 7), and p53 (28, 45). There are also co-activator proteins that do not belong to these two groups, such as ARA70 (85), PGC-1 (60), and the recently-reported RNA co-activator SRA (41). Members of both the SRC-1 family and CBP/p300 family have been shown to possess histone acetyltransferase (HAT) activities (8, 14, 57, 69), suggesting that chromatin remodeling by histone acetylation is an important mechanism involved in transcriptional activation by ligand-bound nuclear receptors.
Interaction of members of the SRC-1 and CBP/p300 families with nuclear receptors occurs through conserved LxxLL (SEQ ID NO:1) motifs (32), which interact with a hydrophobic cleft in the receptor LBD formed as a result of conformational changes mediated by ligand binding (19, 23, 56). In the sequence, x refers to any amino acid. SRC-1/NCoA-1 and GRIP1/TIF2 contain three LxxLL regions or boxes (referred to as LXDs or NR boxes) that differentially interact with nuclear receptors so that different nuclear receptors functionally utilize different LxxLL boxes (19, 52). Thus, ER utilizes the second LxxLL box of SRC-1/NCoA-1 while PR utilizes both the first and second LxxLL boxes for optimal interaction. In contrast, TR and RAR require both the second and third LxxLL boxes for optimal interaction (52). The specificity of receptor recognition by the different LxxLL boxes of SRC-1/NCoA-1 is primarily mediated by eight amino acid residues C-terminal to the LxxLL motif rather than by the two amino acids (xx) within the motif itself. Thus, while members of the SRC-1 family are capable of interacting with many nuclear receptors, the molecular detail of such interactions differs for each receptor in the number or combination of LxxLL boxes utilized as well as in the critical amino acid residues surrounding the LxxLL motifs.
While much has been learned from the study of known co-activators, a number of key mechanistic questions remain to be answered. For example, many nuclear receptors can recognize common DNA elements, (25, 49, 51), while not all are capable of regulating genes containing those elements (20, 47, 65). Thus, how native target genes containing such elements are selectively regulated by specific receptors is a very important but poorly-understood problem. Although the various LxxLL boxes of SRC-1 and GRIP1 show differential receptor preference (19, 52), these co-activators are unlikely to play a primary role in mediating effects that are receptor specific since they appear to interact with all ligand-bound nuclear hormone receptors. Thus, the detailed molecular mechanism(s) underlying receptor-specific regulation of gene expression remains to be elucidated. Whether co-activator(s) might contribute to this specificity is currently unknown.
The present invention discloses an isolated nucleic acid molecule comprising a sequence that encodes a functional NRIF3 nuclear hormone receptor co-activator, where the NRIF3 binds in a ligand dependent manner to thyroid hormone receptor (TR) and retinoid X receptor (RXR), but does not interact with retinoic acid receptor (RAR), vitamin D receptor (VDR), progesterone receptor (PR), glucocorticoid receptor (GR), and estrogen receptor (ER) in a yeast two hybrid assay system or in vitro, or both, and where the nucleic acid encodes a polypeptide that contains an LxxIL (SEQ ID NO:2) module in its C-terminal domain.
A hybridizable nucleic acid, of at least twenty bases, which has a sequence as depicted in SEQ ID NO:3 (FIG. 2) also is contemplated by the present invention.
The present invention also contemplates an isolated functional NRIF3 nuclear hormone receptor co-activator, where the NRIF3 binds in a ligand dependent manner to thyroid hormone receptor (TR) and retinoid X receptor (RXR), but does not interact with retinoic acid receptor (RAR), vitamin D receptor (VDR), progesterone receptor (PR), glucocorticoid receptor (GR), and estrogen receptor (ER) in a yeast two hybrid assay system or in vitro, or both, where the polypeptide contains an LxxIL (SEQ ID NO:2) module in its C-terminal domain.
A method for identifying a compound that modulates NFIR3 interaction with a nuclear hormone receptor is also contemplated by the present invention. The method comprises detecting modulation of the interaction of NFIR3 and TR or RXR in the presence of the compound.